Abstract

Objectives To assess the dose-response relationship, efficacy and safety of tabalumab, a human monoclonal antibody that neutralises
membrane-bound and soluble B-cell activating factor (BAFF), in patients with rheumatoid arthritis (RA) with inadequate response
to methotrexate (MTX).

Methods In this phase 2, 24-week, double-blind, placebo-controlled, dose-ranging study, patients with RA (N=158) on stable MTX were
randomised by Bayesian-adaptive method to receive 1, 3, 10, 30, 60, or 120 mg tabalumab or placebo subcutaneously every 4 weeks
for 24 weeks. The primary objective was to test for a significant dose-response relationship using a statistical model of
the proportion of patients having ≥50% improvement in American College of Rheumatology (ACR) criteria (ACR50) at week 24 (prespecified
α=0.10).

Introduction

Rheumatoid arthritis (RA) affects approximately 1% of the population1 and is characterised by joint inflammation that can lead to joint destruction and systemic complications.2 Currently available biologic therapies selectively target key molecules associated with joint inflammation, but approximately
30% of patients will remain unresponsive to these treatments.3

B-cell activating factor (BAFF) is a tumour necrosis factor (TNF) family ligand that is increased in the sera and synovial
fluid of patients with RA,4–6 and is required for B-cell survival.7 BAFF has two biologically active forms, a soluble and membrane-bound form,8 and induces polyclonal maturation of immature and mature B cells involved in RA pathogenesis.9,10

Tabalumab is a human monoclonal antibody that neutralises soluble and membrane-bound BAFF.11 In a previous study, intravenous tabalumab (30, 60 and 160 mg) reduced RA signs and symptoms in patients with an inadequate
response to methotrexate (MTX-IR); although all doses were effective, no dose-response relationship in American College of
Rheumatology (ACR) scores was observed.12

The current trial, which used Bayesian-adaptive randomisation, explored the dose-response relationship of tabalumab given
subcutaneously once every 4 weeks (Q4W) to patients with active RA receiving stable doses of MTX.

Methods

Patients

Patients were recruited from 64 centres in 12 countries (Argentina, Australia, Chile, Germany, Hungary, India, Mexico, Poland,
Romania, Slovakia, Ukraine and the USA). All patients provided voluntary written informed consent. The study was approved
by local Institutional Review Boards in accordance with the Declaration of Helsinki and applicable laws and regulations.

Patients were aged between 18 and 75 years, were taking MTX (10–25 mg/week) for ≥16 weeks, and met ACR (1987 revised) criteria
for RA.13 Major inclusion criteria included ≥5/28 swollen and ≥5/28 tender joints; ACR functional class I, II, or III; a history of,
or a current, positive rheumatoid factor (RF+) test; a C-reactive protein (CRP) ≥1.2 times upper limit of normal (ULN; 1.0 mg/dl);
and the absence of pregnancy or breast feeding.

Major exclusion criteria included use of any parenteral or oral corticosteroid at >10 mg/day of prednisone or its equivalent
within 4 weeks of baseline; use of any B-cell biotherapies at any time; insufficient response to a TNF inhibitor (TNF-IR;
patients who stopped for reasons other than lack of efficacy were eligible); presence of other autoimmune disorders; a positive
protein derivative test for tuberculosis; or serious bacterial infections within 6 months of enrolment. Study participants
must have discontinued etanercept ≥28 days before baseline, and infliximab, adalimumab, or other biologic TNF inhibitors ≥56 days
before baseline.

Study design

This was a phase 2, 24-week, double-blind, placebo-controlled, dose-ranging study. Following two screening visits, patients
were randomised by Bayesian-adaptive method14 to receive placebo or tabalumab (1, 3, 10, 30, 60, or 120 mg) subcutaneously Q4W for 24 weeks (figure 1A). Patients maintained their prestudy stable dose of MTX and received their other usual medical treatments for RA or concomitant
diseases as allowed by protocol.

Study design and patient disposition. (A) Patients were adaptively randomised by Bayesian method. Over time, the probability
of randomisation to more effective doses increased, resulting in unequal distribution of patients across groups. Study drug
was administered six times by SC injection for 24 weeks. (B) Disposition of 158 randomised and treated patients through week
24. The percentage of patients who withdrew from the study and reasons for discontinuation are also shown for each group.
*One patient in the placebo group was discontinued due to elevated liver enzymes at the sponsor's request. †Two patients in
the 120 mg group were excluded from primary efficacy analyses due sponsor decision to withdraw a site for violation of good
clinical practices. AEs, adverse events, SC, subcutaneous.

Bayesian-adaptive randomisation used accumulating data from the ongoing trial to make progressive adjustments in dose-group
assignments. Over time, these adjustments were expected to provide a more precise estimate of the dose-response relationship.
Computer-generated random treatment assignments were made using an interactive voice-response system (IVRS). The first 35
patients were randomised to 1 of 7 treatment groups in equal number. Once 35 patients were randomised, there was a constant
20% chance of randomisation to placebo and an 80% chance of randomisation to one of the tabalumab doses. A contract research
organisation periodically updated a non-informative, normal, dynamic linear model prior distribution, from which the posterior
distribution of a 24-week, treatment-response model was derived. The posterior distribution was used to adjust the probability
of randomisation to each tabalumab dose. As the study progressed, the probability of assignment to higher doses increased.
Enrolment and randomisation ended when approximately 150 patients had been randomised. These 150 patients provided 80% power
to detect a difference based on simulations using varying responses-to-dose assumptions as well as varying patient accrual
and dropout rates.

Endpoints

The primary endpoint was to test for a significant dose-response relationship based on a statistical model of the proportion
of patients having ≥50% improvement in ACR criteria (ACR50) at week 24. Key secondary efficacy endpoints included the dose-response
relationship based on modelled ACR20 response rates at week 24; ACR20 and ACR50 response rates, change in DAS28, and percent
change in CRP at each visit (weeks 1, 4, 8, 12, 16, 20 and 24); and pharmacokinetic parameters. Safety endpoints included
incidences of adverse events (AEs) and serious adverse events (SAEs), and clinical laboratory test and immunogenicity results.
Pharmacodynamic endpoints included changes over time in serum immunoglobulins (IgM, IgG, IgA), total B cells (CD20), and B-cell
subsets (mature naïve (CD19, IgD+, CD27–), memory (CD19, IgD–, CD27)).

Standard laboratory tests, including chemistry, haematology, urinalysis panels and ECGs were obtained at regular intervals.
Vital signs were taken, and AEs and SAEs were recorded and summarised at each visit.

Statistical methods

All analyses were performed with the intent-to-treat population (all randomised patients who received ≥1 dose of study drug).
Two 120 mg patients were excluded from primary efficacy analyses (site withdrawn due to good clinical practices violation).
For ACR analyses, non-responder imputations (NRIs) were used for patients who discontinued early. For all other efficacy analyses,
a last-observation-carried-forward (LOCF) approach was used.

The primary analysis tested for a significant ACR50 dose-related response over doses ranging from placebo to 120 mg tabalumab
at week 24. The dose-response relationship was tested with a joint test of linear and quadratic dose response (regression
model included terms for dose and dose2) from the likelihood ratio test (α=0.10). The smallest dose achieving ≥95% of maximal efficacy (ED95) and ACR50 response rates corresponding to each dose were estimated from the logistic regression model. This analysis was
repeated for ACR20. The dose-response relationship for DAS28 was performed by Spearman non-parametric correlations with dose
(1-sided, α=0.05).

ACR20 and ACR50 responses were summarised, and Fisher exact tests compared tabalumab observed response rates with placebo
at all timepoints. Modelled response rates were compared using a 1-sided, z test with SE estimated using the delta method.
For DAS28, pairwise comparisons of tabalumab doses versus placebo were performed using contrast statements within an analysis
of covariance (ANCOVA) model with treatment as the fixed factor and baseline as a covariate. Pairwise comparisons were 1-sided
(α=0.05).

Pharmacodynamic analyses were performed by 2-sided comparisons of all tabalumab doses combined versus placebo, using ranked
ANCOVA with the standardised rank outcome variable, treatment as the fixed factor, and the standardised rank baseline value
as a covariate (α=0.10). For CRP and serum immunoglobulins, 2-sided pairwise comparisons of tabalumab dose versus placebo
were performed using contrast statements within ANCOVA (α=0.10).

Tabalumab pharmacokinetic parameters were analysed using a population approach implemented with NONMEM (ICON Development Solutions,
Ellicott City, Maryland, USA). Clearance and distribution volume were characterised as a function of dose and treatment duration.

Safety data were descriptively summarised by treatment. Placebo and tabalumab doses were compared using a χ2 test, or Fisher exact test if χ2 assumptions were violated, for any event.

Results

Patient disposition and characteristics

A total of 142/158 patients (89.9%) completed: 34/36 (94.4%) in the placebo group and 108/122 (88.5%) in all tabalumab groups
combined (figure 1B). The most common reasons for early withdrawal were AEs and patient decision (figure 1B).

Clinical response

A significant (prespecified α=0.10) dose-response relationship was detected for ACR50 (p=0.059) and ACR20 (p=0.044) at week
24 using a regression model. The ED95 was 119.0 mg and 118.5 mg for ACR50 and ACR20, respectively.

Modelled and observed results for ACR50 and ACR20 responders at week 24 are presented in table 2. Using model-estimated data at week 24, only the 120 mg dose had significantly higher ACR50 and ACR20 response rates versus
placebo (table 2).

(A) Values are observed changes in the ACR50 response rate at each treatment visit with the tabalumab dose or placebo; non-responders
imputed. p Values are based on 1-sided Fisher exact test that the tabalumab group has more responders than placebo. *p≤0.05
Versus placebo. (B) Values are observed changes in the ACR20 response rate at each treatment visit with the tabalumab dose
or placebo; non-responders imputed. p Values are based on 1-sided Fisher exact test that the tabalumab group has more responders
than placebo. *p≤0.05 Versus placebo. (C) Time course of LS mean change in DAS28 score at each treatment visit with the tabalumab
dose or placebo. p Values are based on 1-sided pairwise comparison of the tabalumab dose level versus placebo using contrast
statements within an ANCOVA model with treatment as the fixed factor and the baseline value as a covariate. *p≤0.05 Versus
placebo. ACR20, proportion of responders having ≥20% improvement according to American College of Rheumatology (ACR) criteria;
ACR50, proportion of patients having ≥50% improvement according to ACR criteria; ANCOVA, analysis of covariance; DAS28, Disease
Activity Score based on 28-joint count; LOCF, last-observation-carried-forward, LS mean, least squares mean; SC, subcutaneous.

At baseline, DAS28 scores were similar across groups (table 1). DAS28 score significantly improved from baseline with 120 mg versus placebo at week 24 (table 2); this improvement was also observed at earlier timepoints (figure 2C). No other dose showed a significant DAS28 improvement.

At week 24, tender joint counts, swollen joint counts, and patient's assessments of disease activity and pain were similar
across groups, whereas physician's assessment of disease activity was significantly reduced with 120 mg (table 3). Mean CRP was similar between placebo and tabalumab groups at week 24. In some patients, elevated CRP at screening was no
longer elevated at baseline. A posthoc analysis of patients with baseline CRP>ULN was conducted by treatment group (placebo,
1, 3, 10, 30, 60 and 120 mg). For these groups, baseline CRP was 38.1, 27.0, 45.0, 38.4, 30.5, 37.4 and 30.2 mg/dl, and mean
CRP (LOCF) at week 24 was 16.8, 8.1, −8.8, 21.8, 46.2, 38.9 and 38.5 mg/dl, respectively.

Pharmacokinetics

Serum tabalumab concentrations demonstrated non-linear elimination. The time to reach maximum concentration following subcutaneous
injection at steady state (Tmax,ss) and the half-life over the 4-week dosing interval (t1/2,tau) increased with increasing dose. At 120 mg, Tmax,ss and t1/2,tau were 5.0 and 21.6 days, respectively.

Mean IgM and IgA levels tended to be lower than baseline for all tabalumab groups at weeks 16 and 24. For IgM, this difference
was statistically significant at week 16 with the 30 mg (–0.15±0.40 g/l, p=0.016), 60 mg (−0.24±0.27 g/l, p=0.028), and 120
mg (−0.17±0.32 g/l, p=0.005) versus placebo (0.02±0.39 g/l), and at week 24 with 30 mg (−0.08±0.53 g/l, p=0.023), 60 mg (−0.30±0.30 g/l,
p=0.004), and 120 mg (−0.09±0.38 g/l, p=0.017) versus placebo (0.02±0.32 g/l). For IgA, this difference was statistically
significant at week 16, with 120 mg versus placebo (−0.32±0.42 vs −0.12±0.50 g/l, p=0.040), and at week 24 with 60 mg (−0.42±0.45 g/l,
p=0.025) and 120 mg (−0.25±0.63 g/l, p=0.030) versus placebo (−0.12±0.48 g/l). Mean IgG levels were not significantly different
from placebo with any tabalumab dose at any timepoint. There was no association between changes in serum immunoglobulin levels
and occurrence of infection or other AEs. There was no correlation between clinical response, B-cell counts, or serum immunoglobulin
levels.

Safety

The frequency of treatment-emergent adverse events (TEAEs) was similar across tabalumab doses (range 50–69%, p=0.884) (table 4). The majority of TEAEs were mild or moderate in severity with no obvious trends in the nature or frequency by dose. The
most frequently reported TEAEs were injection-site pain and upper respiratory tract infection (table 4). Five patients discontinued due to an AE, all of whom were treated with tabalumab 1 mg (hemiplegia), 3 mg (prolonged QT
and RA worsening (exacerbation/flare)), or 120 mg (diverticulitis and RA worsening). The incidence of SAEs was 13.9% (5/36
patients) with placebo and ranged from 3.8% (1/26 patients) with 120 mg to 16.7% (3/18 patients) with 30 mg (table 4). RA worsening was the only SAE reported in >1 patient (n=2, placebo). No patients died during the study.

Infection was reported at a higher incidence with tabalumab (30.3% (37/122 patients)) than placebo (19.4% (7/36 patients)),
but did not increase with higher doses. The lowest incidence of infection was observed with 120 mg (11.5%). Serious infections
were reported in three (2.5%) tabalumab-treated patients. One patient discontinued due to a serious infectious event of H1N1
influenza pneumonia 24 days after a single 30 mg dose.

No clinically relevant differences or trends were identified in vital signs, ECG, or chemistry, haematology and urinalysis
panels. The percentage of patients with abnormal laboratory values was comparable across groups, with no apparent trends.

Immunogenicity

At 24 weeks, two tabalumab-treated patients had treatment-emergent antitabalumab antibodies (TEAb (fourfold increase from
baseline); 1 mg and 120 mg group). One patient seroconverted at week 4, the other at week 8, and TEAb persisted to week 24.
None were neutralising. Two additional patients (1 mg and 10 mg group) and 2 placebo patients had transient TEAb detected
at a single sampling. One placebo patient had neutralising antibodies at week 24. The presence of TEAb did not appear to have
an effect on ACR response. None of the tabalumab-treated patients who tested positive for antibodies experienced an SAE or
serious infection, nor did they discontinue due to lack of efficacy. There was no discernible reduction in tabalumab serum
concentrations in the presence of antitabalumab antibodies and no dose-related trends.

Discussion

In the current study, which used Bayesian-adaptive randomisation, a statistically significant dose-response relationship was
detected using regression models of ACR50 and ACR20 response rates at 24 weeks for tabalumab, administered monthly by subcutaneous
injection over a 1 mg to 120 mg dose range. Significant efficacy versus placebo was detected with the 120 mg dose for ACR50
(by regression analysis only), ACR20, and DAS28 at week 24.

The principles and potential utility of B-cell depletion in RA treatment have been recognised since 2001. 16 Subsequent work resulted in the approval of rituximab for RA treatment in TNF-IR patients.17 The concept of effective RA treatment through B-cell inhibition (without profound depletion) has been explored with limited
success. Belimumab, a monoclonal antibody that neutralises soluble BAFF, was studied but not developed as an RA treatment
after modest phase 2 results.18,19 Atacicept, a fusion protein targeting BAFF and APRIL (a proliferation–inducing ligand), failed to achieve clinical benefits
in two RA trials.20,21

Overall, no unexpected safety signal was detected in this relatively small study; tabalumab had a consistent safety profile
across dose groups and placebo. Infectious events were more frequent with tabalumab than placebo, although the frequency did
not increase with higher doses. Mean IgM and IgA levels tended to be lower than baseline in all tabalumab groups in the later
weeks of the study; changes from baseline were not associated with increases in AEs or infectious AEs.

Within 1 week of the first injection, CD20 B cells and CD19 mature, naïve cells transiently increased with all tabalumab doses
before decreasing back to baseline levels or below without profound reductions. Early increases in B cells with subsequent
decreases have been observed with other BAFF-targeted therapies, such as briobacept22 and atacicept.23 No correlations were observed between B-cell changes and clinical efficacy or safety with tabalumab.

The following limitations of this study should be considered. Bayesian-adaptive randomisation was intended to estimate the
dose-response relationship more precisely and to allocate patients to more effective dosing. However, patients were enrolled
faster than planned, and updates to randomisation probabilities were not frequent enough. As a result, a higher percentage
of patients were assigned to placebo or very low dose treatments. Despite this, a dose-response relationship was detected.
Additionally, this was a short-term study with small treatment arms, and ACR50, rather than ACR20, was the primary endpoint.
This study enrolled patients with active RA who were taking MTX, but not patients who previously failed TNF inhibitors or
other biologic RA treatments, and only included RF+ patients. These findings cannot be generalised to patients on other background
RA medications, with a history of exposure to a larger repertoire of agents, or who are seronegative. Despite these limitations,
tabalumab demonstrated efficacy in MTX-IR patients.

In the present study, subcutaneous 120 mg tabalumab appeared to reduce RA signs and symptoms in patients taking concomitant
MTX. Overall, based on a limited number of exposures in this phase 2 study, tabalumab had no unexpected safety signals. After
this study was completed, phase 3 clinical trials were undertaken using tabalumab in patients with RA. These trials were recently
discontinued after interim analyses provided results that did not meet efficacy expectations.24,25 No safety concerns were noted.

Acknowledgments

The authors would like to thank all the participating investigators. The authors would also like to thank Kelly Guerrettaz
(PharmaNet/i3, an inVentiv Health Company) for writing assistance, and Pam Boltz (PharmaNet/i3, an inVentiv Health Company),
Teri Tucker (PharmaNet/i3, an inVentiv Health Company), and Dr Pamela W Anderson (Eli Lilly and Company) for editorial assistance.

Footnotes

Handling editor Tore K Kvien

Contributors MCG contributed to the study design, was involved in the data analysis, and reviewed/edited the manuscript. EL participated
as an investigator and reviewed/edited the manuscript. JS, MV, and DD contributed to the data analysis and reviewed/edited
the manuscript. GS contributed to the study design. OB, SM, and P-YB contributed to the study design, implementation, and
analysis, and reviewed/edited the manuscript.

Lilly discontinues one of three phase 3 rheumatoid arthritis registration studies for tabalumab—FLEX-M study did notmeet efficacy expectations in an interim futility analysis; discontinuation not based on safety concerns. Eli Lilly and Company. 13 December 2012.

Lilly discontinues phase 3 rheumatoid arthritis program for tabalumab based on efficacy results—decision not based on safety
concerns; phase 3 lupus program continues as planned. Eli Lilly and Company. 7 February 2013.